1. Field of the Invention
The present invention relates to polyhydroxyalkanoate (hereinafter referred to as xe2x80x9cPHAxe2x80x9d) biosynthesis-related genes for PHA synthase, xcex2-ketothiolase and acetoacetyl-CoA reductase, derived from Alcaligenes latus, their amino acid sequences, a recombinant plasmid carrying these genes, and a method for mass producing PHA using these genes.
2. Description of the Prior Art
Petroleum synthetic plastics are so durable that they are not degraded in usual conditions at all. Because the production amount of the petroleum synthetic plastics increases each year, the environmental pollution ascribed to petroleum synthetic plastics wastes are now a big social problem. To solve the problem of non-degradable plastics, active research and development efforts have been and continued to be directed to biodegradable polymers all over the world.
Biodegradable polymers are the high molecular weight materials that are completely degraded under natural conditions after a period of time. Many biodegradable polymers have been developed. Of them, PHA, a natural polyester which is synthesized and accumulated by microorganisms, is of particular interest because it is superior in biodegradability as well as shows physical properties similar to those of the synthetic plastics in current use (Anderson A. J. and Dawes, E. A., Microbiol. Rev., 1990, 54, 450-472; Lee, S. Y., Biotechnol. Bioeng., 49:1-14,1996; Lee, S. Y., Trends Biotechnol., 14:431 -438, 1996).
In detail, PHA is an organic reserve material, which can provide an intracellular store of carbon or energy, usually found in Pseudomonas, Alcaligenes, Azotobacter, and Bacillus spp.,etc. It is detectable as granular cytoplasmic inclusions. As a general rule, the cellular content of the reserve material is relatively low in actively growing cells: They accumulate massively when cells are limited in nitrogen, phosphorous, sulfur, oxygen, etc., but still have carbon and energy available. This reserve material was first found in Bacillus megaterium by Lemoigne in 1925 (Lemoigne, M., Bull. Soc. Chem. Biol., 8:770-782, 1926). Since then, its chemical and physical properties have been extensively researched. Poly(3-hydroxybutyrate) is the most widely, and first known PHA.
According to the number of carbon atoms and the substituents in hydroxyalkanoate, many PHAs were reported. In general, PHAs are divided into two classes; short-chain-length PHAs(SCL PHAs) and medium-chain-length PHAs(MCL PHAs)
SCL PHAs include poly-xcex2-hydroxypropionic acid, poly-xcex2-hydroxybutyric acid, and poly-xcex2-hydroxyvaleric acid, which are produced by Alcaligenes eutrophus, Azotobacter vinelandii, methylotrophs, etc. SCL PHAs are widely used due to their similar properties to polypropylene, a kind of chemically synthesized plastics.
MCL PHAs, composed of 3 to 9 more carbon atoms than SCL PHAs, are produced by Pseudomonas spp., by using alkane, 1-alkene, C6xcx9cC12 alkanoic acids as a carbon.
Since early the 1960s, it was recognized that PHA could work like thermoplastic polymers. Thereafter, attracting a great attention, many types of PHA copolymers were synthesized, which are superior in mechanical properties as well as in biodegradability. By virtue of these advantages and owing to the environmental pollution aggravated by petroleum synthetic polymer wastes, PHA is now actively researched and developed as an alternative for plastics over the world. In addition, biocompatibility and bioabsorptivity allow PHA to be used in a variety of fields, as materials for agriculture, medicinal care, drug transfer system, and package, and as precursors for fine chemical products (Holmes, P. A. in Developments in crystalline polymers. 1-65, 1988).
Taking advantage of various bacteria, molecular biological research has revealed that there are four different biosynthetic pathway for PHA (Steinbuchel, A. in Biomaterials: novel materials from biological sources, 215-262, 1991). For example, for Alcaligenes eutrophus, the most widely known bacteria, xcex2-ketothiolase, acetoacetyl-CoA reductase and polyhydroxyalkanoate synthase (PHA synthase) are known to be involved in the biosynthesis of PHA (People, O. P. and Shinskey, A. J., J. Biol Chem., 264: 15298-15303, 1989; Schubert, P., Steinbuchel, A. and Schlegel, H. G., J. Bacteriol., 170:5837-5847, 1988; Slater, S. C., Voige, W. H. and Dennis, D. E., J. Bacteriol., 170:4431-4436, 1988).
A concrete biosynthetic pathway of PHA in Alcaligenes eutrophus, gram negative bacteria, is as follows. Between two molecules of acetyl-CoA, a carbon-carbon bond forms in the presence of xcex2-ketothiolase, the product of gene phbA , according to a biological Claisen condensation. The acetoacetyl-CoA thus formed is converted into D(xe2x88x92)-xcex2-hydroxybutyryl-CoA by the stereoselective reduction of NADPH-dependent acetoacetyl-CoA reductase, the product of gene phbB. Finally, D(xe2x88x92)-xcex2-hydroxybutyryl-CoA is polymerized via ester bond by PHA synthase, the product of gene phbC.
In order to clone the genes which pertain to the biosynthesis of PHA in other bacteria than Alcaligenes eutrophus, much effort has been made. That is, the comprehension of the biosynthesis of PHA in bacteria makes it possible efficient production of PHA, versatility of substrates, synthesis of new PHA, and development of biopolymers similar to PHA. Further, recombinant strains which are obtained by utilizing the PHA biosynthesis-related genes can synthesize various PHAs at high efficiencies, resulting in a scientific and industrial significance (Lee, S. Y., Trends Biotechnol., 14:431-438, 1996).
Strain Alcaligenes latus is reported to be so superior in the production of PHA that it accumulates PHA in cells at a proportion of around 90%. Also, Alcaligenes latus as the advantage in that it grows fast and uses inexpensive substrates as carbon sources (Wang, F. and Lee, S. Y., Appl. Environ. Microbiol., 63:3703-3706, 1997). Unlike Alcaligenes eutrophus, Alcaligenes latus accumulates PHAs while they are growing. Thus, Alcaligenes latus can mass-produce PHA by one-step culture although the amount is low relative to that upon Alcaligenes eutrophus. 
The use of Alcaligenes latus to produce PHA began in earnest in the mid-1980s by Chemie Linz AG, Austria. Biotechnologishe forchungesellschaft mbH, Austria, developed a process in which a one-step culture of strain btF-96, a mutant strain of Alcaligenes latus., produces PHA, asserting that one ton of PHA is obtained from a 15 m3 ferinentor per week (Hrabak, O., FEMS Microbial. Rev., 103:251-256, 1992). Alcaligenes latus also produces poly(3-hydroxybutyrate/3-hydroxypropionate) as well as poly(3-hydroxybutyrate/4-hydroxypropionate) in a medium containing disaccharides as carbon source by addition of 3-hydroxypropionate and xcex3-butyrolactone (Hiramitsu, M., Koyama, N., and Doi, Y., Biotechnol. Leit., 15:461-464, 1993).
PHA can be produced by chemical process as well as biological process. However, Commercially favorable production scale of PHA is possible only by biological process. Since the production cost of PHA is much higher than those of other commercially available synthetic polymers, new technologies are required to reduce the production cost of PHA. Particularly, recombinant DNA technology gives a great contribution to the development and modification of novel strains, showing the production of novel polymers, utility of low-priced substrate, high efficiency of production, and facility in separation and purification. In order to develop such recombinant strains, first of all, it is necessary to understand the enzymes involved in the biosynthetic pathway for PHA.
In order to mass-produce biodegradable, natural PHA and its copolymers, the inventors have cloned genes for polyhydroxyalkanoate synthase, xcex2-ketothiolase, and acetoacetyl-CoA reductase, and determined amino acid sequences and gene sequences. They have made expression vectors carrying the above genes and transformants, whereby polyhydroxyalkanoate can be produced and accumulated.